Glioblastomas (GBM) are some bad prognosis brain tumors despite a conventional treatment associating surgical resection and subsequent radio-chemotherapy. Among these heterogeneous tumors, a subpopulation of chemo- and radioresistant GBM stem-like cells appears to be involved in the systematic GBM recurrence. Moreover, recent studies showed that differentiated tumor cells may have the ability to dedifferentiate and acquire a stem-like phenotype, a phenomenon also called plasticity, in response to microenvironment stresses such as hypoxia. We hypothesized that GBM cells could be subjected to a similar dedifferentiation process after ionizing radiations (IRs), then supporting the GBM rapid recurrence after radiotherapy. In the present study we demonstrated that subtoxic IR exposure of differentiated GBM cells isolated from patient resections potentiated the long-term reacquisition of stem-associated properties such as the ability to generate primary and secondary neurospheres, the expression of stemness markers and an increased tumorigenicity. We also identified during this process an upregulation of the anti-apoptotic protein survivin and we showed that its specific downregulation led to the blockade of the IR-induced plasticity. Altogether, these results demonstrated that irradiation could regulate GBM cell dedifferentiation via a survivin-dependent pathway. Targeting the mechanisms associated with IR-induced plasticity will likely contribute to the development of some innovating pharmacological strategies for an improved radiosensitization of these aggressive brain cancers.

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks repair. Ku is also expressed on the cell surface of different types of cells where its function remains poorly understood. From a yeast two-hybrid screen, we have identified a specific interaction between the core region of Ku80 and the hemopexin domain of metalloproteinase 9 (MMP-9), a key enzyme involved in the degradation of extracellular matrix (ECM) components. Ku associates with MMP-9 on the surface of leukemic cells as demonstrated by co-immunoprecipitation experiments in membrane extracts and double-label immunofluorescence studies. In normal and tumoral migratory cells, Ku80 and MMP-9 colocalize at the periphery of leading edge of cells and cellular invasion of collagen IV matrices was blocked by antibodies directed against Ku70 or Ku80 subunits as well as by Ku80-specific antisense oligonucleotides. Our results indicate that Ku and MMP-9 interact at the cell membrane of highly invasive hematopoietic cells of normal and tumoral origin and document the unexpected importance of the membrane-associated form of Ku in the regulation of ECM remodelling.

Glioblastomas are malignant brain tumors with dismal prognosis despite standard treatment with surgery and radio/chemotherapy. These tumors are defined by an important cellular heterogeneity and notably contain a particular subpopulation of Glioblastoma-initiating cells, which recapitulate the heterogeneity of the original Glioblastoma. In order to classify these heterogeneous tumors, genomic profiling has also been undertaken to classify these heterogeneous tumors into several subtypes. Current research focuses on developing therapies, which could take into account this cellular and genomic heterogeneity. Among these targets, integrins are the subject of numerous studies since these extracellular matrix transmembrane receptors notably controls tumor invasion and progression. Moreover, some of these integrins are considered as membrane markers for the Glioblastoma-initiating cells subpopulation. We reviewed here integrin expression according to glioblastoma molecular subtypes and cell heterogeneity. We discussed their roles in glioblastoma invasion, angiogenesis, therapeutic resistance, stemness and microenvironment modulations, and provide an overview of clinical trials investigating integrins in glioblastomas. This review highlights that specific integrins could be identified as selective glioblastoma cells markers and that their targeting represents new diagnostic and/or therapeutic strategies.

Integrins are extracellular matrix receptors involved in tumour invasion and angiogenesis. Although there is evidence that inhibiting integrins might enhance the efficiency of radiotherapy, little is known about the exact mechanisms involved in the integrin-dependent modulation of tumor radiosensitivity. The purpose of this study was to investigate the role of avb3 and avb5 integrins in glioblastoma cell radioresistance and overall to decipher the downstream biological pathways. We first demonstrated that silencing avb3 and avb5 integrins with specific siRNAs significantly reduced the survival after irradiation of 2 glioblastoma cell lines: U87 and SF763. We then showed that integrin activity and integrin signalling pathways controlled the glioma cell radiosensitivity. This regulation of glioma cell response to ionising radiation was mediated through the integrin-linked kinase, ILK, and the small GTPase, RhoB, by two mechanisms. The first one, independent of ILK, consists in the regulation of the intracellular level of RhoB by avb3 or avb5 integrin. The second pathway involved in cell radiosensitivity consists in RhoB activation by ionising radiation through ILK. Furthermore, we demonstrated that the avb3/avb5 integrins/ILK/ RhoB pathway controlled the glioma cells radiosensitivity by regulating radiation-induced mitotic cell death. This work identifies a new biological pathway controlling glioblastoma cells radioresistance, activated from the membrane through avb3 and/or avb5 integrins via ILK and RhoB. Our results are clues that downstream effectors of avb3 and avb5 integrins as ILK and RhoB might also be promising candidate targets for improving the efficiency of radiotherapy and thus the clinical outcome of patients with glioblastoma. ' 2008 Wiley-Liss, Inc.Key words: glioblastoma; radiosensitivity; integrin; ILK; RhoB Despite recent advances in diagnostic imaging, neurosurgical techniques, radiation therapy and chemotherapy, 1 glioblastoma, one of the most common primary brain tumours, is still associated with a dismal prognosis. Almost all of the patients will die of a relapse in the radiation field as a result of low tumour sensitivity to ionising radiation. The low tumour sensitivity to radiotherapy is not only due to the modulation of different biological signal transduction pathways in tumour cells but also due to a cross-talk between the tumour cells and their microenvironment. Our previous results have shown that factors controlling the microenvironment, such as basic fibroblast factor (FGF-2), modulate the tumour cell radioresistance via the small GTPase RhoB which itself also regulates angiogenesis via metalloproteinase 2 (MMP2) and hypoxia in the glioblastoma U87 cell model. Cell adhesion to extracellular matrix (ECM) is known to confer a cell-adhesion-mediated chemotherapeutic drug resistance for hematopoietic and solid tumours.9 Among integrins, the b1 integrin has been largely involved in cell survival after a genotoxic injury by modulating DNA repair 10 or protecting cells from apoptosis. 11Fe...

The presence of hypoxic areas in glioblastoma is an important determinant in tumor response to therapy and, in particular, to radiotherapy. Here we have explored the involvement of integrins, up to now known as regulators of angiogenesis and invasion, in the regulation of tumor hypoxia driven from the tumor cell. We first show that hypoxia induces the recruitment of A v B 3 and A v B 5 integrins to the cellular membrane of U87 and SF763 glioblastoma cells, thereby activating the focal adhesion kinase (FAK). We then show that inhibiting A v B 3 or A v B 5 integrins in hypoxic cells with a specific inhibitor or with siRNA decreases the hypoxia-inducible factor 1A (HIF-1A) intracellular level. This integrin-dependent regulation of HIF-1A is mediated through the regulation of FAK, which in turn activates the small GTPase RhoB, leading to the inhibition of GSK3-B. Furthermore, silencing this pathway in glioma cells of established xenografts dramatically reduces glioma hypoxia, associated with a significant decrease in vessel density. Our present results unravel a new mechanism of hypoxia regulation by establishing the existence of an A v B 3 /A v B 5 integrin-dependent loop of hypoxia autoregulation in glioma. Targeting this hypoxia loop may be crucial to optimizing radiotherapy efficiency.

Hypoxia is a crucial factor in tumor aggressiveness and resistance to treatment, particularly in glioma. Our previous results have shown that inhibiting the small GTPase RhoB increased oxygenation of U87 human glioblastoma xenografts, in part, by regulating angiogenesis. We investigated here whether RhoB might also control a signaling pathway that would permit glioma cells to adapt to hypoxia. We first showed that silencing RhoB with siRNA induced degradation and inhibition of the transcriptional activity of the hypoxiainducible factor by the proteasome in U87 hypoxic cells. This RhoB-dependent degradation of hypoxia-inducible factor-1A in hypoxic conditions was mediated by the Akt/glycogen synthase kinase-3B pathway. While investigating how hypoxia could activate this signaling pathway, using the GST-Rhotekin RBD pulldown assay, we showed the early activation of RhoB by reactive oxygen species under hypoxic conditions and, subsequently, its participation in the ensuing cellular adaptation to hypoxia. Overall, therefore, our results have not only highlighted a new signaling pathway for hypoxia controlled by the small GTPase RhoB, but they also strongly implicate RhoB as a potentially important therapeutic target for decreasing tumor hypoxia. (Cancer Res 2006; 66(1): 482-9)

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c Unlike other Rho GTPases, RhoB is rapidly induced by DNA damage, and its expression level decreases during cancer progression. Because inefficient repair of DNA double-strand breaks (DSBs) can lead to cancer, we investigated whether camptothecin, an anticancer drug that produces DSBs, induces RhoB expression and examined its role in the camptothecin-induced DNA damage response. We show that in camptothecin-treated cells, DSBs induce RhoB expression by a mechanism that depends notably on Chk2 and its substrate HuR, which binds to RhoB mRNA and protects it against degradation. RhoB-deficient cells fail to dephosphorylate ␥H2AX following camptothecin removal and show reduced efficiency of DSB repair by homologous recombination. These cells also show decreased activity of protein phosphatase 2A (PP2A), a phosphatase for ␥H2AX and other DNA damage and repair proteins. Thus, we propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of ␥H2AX and DSB repair. Finally, we show that RhoB-deficient cells accumulate endogenous ␥H2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression.